Use of artemisinin for treating tumors induced by oncogenic viruses and for treating viral infections

ABSTRACT

In certain aspects, the invention relates to methods of treating proliferative cervical disorders (such as cervical cancer and cervical dysplasia) and treating virus infections by administering artemisinin-related compounds. In certain aspects, the invention relates to methods of treating a tumor induced by an oncogenic virus, methods of killing or inhibiting a squamous cell carcinoma, and methods of inhibiting the replication of a virus, by administering artemisinin-related compounds.

BACKGROUND

Despite the advent of the Papanicolaou (Pap) smear, cervical cancers andpre-cancers remain important health problems for women, especially inpoorly monitored women in the United States and developing countries.Worldwide, 250,000 women die annually from this cancer. Cervicaldysplasia is a premalignant or precancerous change to the cervicalcells, and can progress into cervical cancer without treatment.

The major risk factor for cervical cancer is infection by humanpapillomavirus (HPV). Papillomavirus infections are responsible for 99%of cervical cancers in women, as well as the majority of anorectalsquamous cell carcinomas. In addition, papillomaviruses are found insquamous and basal cell carcinomas of the skin as well as in squamouscarcinoma cells of the mouth, oropharynx, and larynx. Thepapillomaviruses also induce many benign tumors including genital wartsand common warts of the hands and feet. They also induce laryngealpapillomas of children and adults. Currently, there are nopharmaceutical therapies available for the treatment of humanpapillomavirus (HPV) infections and the accompanying tumors that theyinduce.

Clinical trials have evaluated the injection of interferon intopapillomavirus lesions and have shown some effect. However, the viralinfections recur immediately after withdrawal of the interferon. Topicalapplications of other compounds such as 5-fluorouracil (5FU) andpodophyllotoxin are toxic and kill both infected and normal cells. Thesetreatments are not highly effective and do not specifically target thetumor cells. A recent therapy which is in trials is the use of IonicContra Viral Therapy (ICVT), which was developed by Henderson Marley(see, e.g., WO 01/49300, WO 01/49242, WO 01/66100, WO 02/24207). Sinceantiviral therapies are not available for papillomavirus infections, thecurrent clinical approach is to develop a vaccine that will preventinfection. Vaccines offer great promise and animal studies have foundthem to be highly protective. However, among the 100 types ofpapillomaviruses that infect humans, there are at least 5 types thatinduce cervical cancer and it will be necessary to develop a multivalentvaccine in order to prevent this cancer in women. Current trials areevaluating only a monovalent vaccine against HPV type 16 and thesetrials will not be completed for several years. It will then benecessary to develop techniques to express the capsid proteins of theother HPV types, which is not necessarily a routine procedure.

For many years, virus diseases have been considered as intractable toselective antiviral chemotherapy because the replicative cycle of thevirus was assumed to be too closely interwoven with normal cellmetabolism and any attempt to suppress virus reproduction would alsokill (or severely harm) uninfected cells as well. Clearly, there is aneed for additional approaches to treating conditions such as virusinfections and cancers (e.g., cervical cancer) resulting from virusinfection, which are significant public health problem.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating infection caused byviruses including human papillomavirus (HPV), human T-lymphotropic virustype I (HTLV-1), herpes virus (e.g., Epstein-Barr virus (EBV),cytomegalovirus (CMV)), SV40-like virus, hepatitis virus, humanimmunodeficiency virus (HIV), adenovirus, and influenza virus, as wellas treatment of cervical disorders associated with virus infection(e.g., cervical cancer and cervical dysplasia), through theadministration of artemisinin and/or artemisinin derivatives. Thepresent invention particularly relates to methods of selectively killingor inhibiting growth of cells, such as premalignant (precancerous) andmalignant (cancerous) cells by administering artemisinin and/or anartemisinin derivative (one or more derivatives).

In one embodiment, the invention provides a method of treating anindividual suffering from a virus infection. An individual (patient orsubject) suffering from a virus infection is treated by administering tothe individual a therapeutically effective amount of anartemisinin-related compound. As used throughout the application, theterm “artemisinin-related compound” includes both artemisinin andartemisinin derivatives (analogs). The viral infection may be caused bya virus such as human papillomavirus (HPV), human T-lymphotropic virustype I (HTLV-1), herpes virus (e.g., Epstein-Barr virus (EBV),cytomegalovirus (CMV)), SV40-like virus, hepatitis virus, humanimmunodeficiency virus (HIV), adenovirus, and influenza virus.

The method of the present invention can be used to treat bothpremalignant and malignant cervical lesions due to papillomavirus. Inanother embodiment, the invention provides a method of treating anindividual suffering from a proliferative cervical disorder. In thisembodiment, an individual suffering from a proliferative cervicaldisorder is treated by administering to the individual a therapeuticallyeffective amount of an artemisinin-related compound. As used herein, theterm “proliferative cervical disorder” includes cervical cancer andcervical precancer (e.g., cervical dysplasia). The proliferativecervical disorder may be associated with papillomavirus infection.

Artemisinin derivatives include, but are not limited to,dihydroartemisinin, artemether, arteether, artesunate, artelinic acid,and dihydroartemisinin propyl carbonate. An artemisinin-related compoundmay be administered to the individual by a variety of routes, forexample, orally, topically, parenterally, intravaginally, systemically,intramuscularly, rectally or intravenously. In certain embodiments, anartemisinin-related compound is formulated with a pharmaceuticalcarrier.

In some embodiments, artemisinin or an artemisinin derivative iscombined with other anti-viral or anti-cancer therapies, such asadministration of an anti-viral or anti-cancer agent, radiation therapy,phototherapy or immunotherapy. The anti-viral or anti-cancer agent canbe administered with an artemisinin-related compound either in the sameformulation or in separate formulations, to enhance treatment. In theseembodiments, the artemisinin-related compound and the other therapiescan be administered at the same time (simultaneously) or at separatetimes (sequentially), provided that they are administered in such amanner and sufficiently close in time to have the desired effect.

In another embodiment, the invention provides a method of killing orinhibiting growth of cells that are infected by human papillomavirus,such as cervical cancer cells, anorectal squamous cancer cells, squamouscarcinoma cells of the skin, basal carcinoma cells of the skin, andsquamous carcinoma cells of the mouth, oropharynx, and larynx. Squamouscell carcinomas of the head and neck, esophagus, trachea, and bronchi(some of which contain HPV) are also potential targets. The infectedcells are contacted with an artemisinin-related compound in a sufficientamount to kill or inhibit growth of infected cells. In this embodiment,a therapeutically effective amount of an artemisinin-related compound isadministered to an individual in need of treatment, e.g., for treatmentof cervical cancer, anorectal cancer, squamous or basal cell skincancer, and squamous carcinoma of the mouth, oropharynx, and larynx. Theartemisinin-related compound is administered by a route appropriate forits delivery to the site(s) at which treatment is needed (e.g.,topically, intravaginally, rectally, orally, systemically,intramuscularly or intravenously) in an amount sufficient to kill orinhibit growth of cells infected by human papillomavirus.

In another embodiment, the invention provides a method of killing orinhibiting growth of cells that are infected by oncogenic viruses suchas HPV, HTLV-1, herpes virus (e.g., EBV or CMV), SV40-like viruses,hepatitis virus, or adenovirus. In addition, HIV, and influenza virusare potential targets. The infected cells are contacted with anartemisinin-related compound in a sufficient amount to kill or inhibitgrowth of the infected cell. In this embodiment, a therapeuticallyeffective amount of an artemisinin-related compound is administered toan individual in need of treatment of infection by HPV, HTLV-1, herpesvirus (e.g., EBV or CMV), SV40-like virus, hepatitis virus, HIV,adenovirus or influenza virus, by a route which results in (isappropriate for) delivery of an amount sufficient to kill or inhibitgrowth of infected cells to the sites at which treatment is needed.

In another embodiment, the invention provides a method of treating anindividual infected with human papillomavirus (HPV). In this embodiment,a therapeutically effective amount of an artemisinin-related compound isadministered to the individual by a route appropriate for delivery of anamount sufficient to kill or inhibit growth of infected cells to thesite(s) at which treatment is needed. This embodiment is useful to treata variety of conditions in which an individual is infected with HPV,such as conditions in which the cells to be killed or inhibited arecervical cancer cells, anorectal squamous cancer cells, squamouscarcinoma cells of the skin, basal carcinoma cells of the skin, andsquamous carcinoma cells of the mouth, oropharynx, and larynx. Squamouscell carcinomas of the head and neck, esophagus, trachea, and bronchiare also potential targets

In another embodiment, the invention provides a method of treating anindividual infected with a virus such as HPV, HTLV-1, herpes virus(e.g., EBV or CMV), SV40-like virus, hepatitis virus, HIV, adenovirus orinfluenza virus. In this embodiment, a therapeutically effective amountof an artemisinin-related compound is administered to an individual inneed of treatment of infection by HPV, HTLV-1, herpes virus (e.g., EBVor CMV), SV40-like virus, hepatitis virus, HIV, adenovirus or influenzavirus, by a route which results in (is appropriate for) delivery of anamount sufficient to kill or inhibit growth of infected cells to thesites at which treatment is needed.

In another embodiment, the invention provides a method of treating acondition caused by human papillomavirus in an individual. In thisembodiment, a therapeutically effective amount of an artemisinin-relatedcompound is administered to the individual by a route appropriate fordelivery of an amount sufficient to kill or inhibit growth of infectedcells to the site(s) at which treatment is needed. Conditions caused byHPV that may be treated by the subject methods include, but are notlimited to, cervical cancer, anorectal squamous cancer, squamouscarcinoma or basal carcinoma of the skin, and squamous carcinoma of themouth, oropharynx, larynx, head and neck, esophagus, trachea, andbronchi.

In another embodiment, the invention provides a method of treating acondition caused by a virus in an individual. Conditions caused by avirus, such as HPV, HTLV-1, herpes virus (e.g., EBV or CMV), SV40-likevirus, hepatitis virus, HIV, adenovirus, or influenza virus, can betreated by the method of the present invention. In this embodiment, atherapeutically effective amount of an artemisinin-related compound isadministered to the individual by a route appropriate for delivery of anamount sufficient to kill or inhibit growth of infected cells to thesite(s) at which treatment is needed. Conditions caused by such virusesthat may be treated by the subject methods include, but are not limitedto, cancers, such as cervical cancer, anorectal squamous cancer,squamous carcinoma or basal carcinoma of the skin, and squamouscarcinoma of the mouth, oropharynx, larynx. Viruses which have theability to induce tumors in man or animals are referred to as“oncogenic” viruses. That is, they target oncogenic viruses. Theyinclude, but are not limited to, HPV, HTLV-1, herpes virus (e.g., EBV orCMV), SV40-like virus, hepatitis virus, and adenovirus.Artemisinin-related compounds can also be used to inhibit non-oncogenicviruses, such as HIV and influenza virus.

In another embodiment, the invention provides a method of treatingnon-malignant papillomavirus infections, such as benign tumors in anindividual, e.g., laryngeal papillomas, genital papillomas (warts), andcommon warts of hands and feet. The infected cells are contacted with anartemisinin-related compound in an amount sufficient to kill or inhibitgrowth of the cells. For example, a therapeutically effective amount ofan artemisinim-related compound is administered to an individual by aroute which results in delivery of the compound to target site(s) (e.g.,laryngeal tissue, genital warts or common warts of hands and feet) atwhich infected cells are to be killed or inhibited.

In another embodiment, the invention provides a method of treating anindividual suffering from a tumor induced by an oncogenic virus. In thisembodiment, an individual suffering from a tumor which is induced by anoncogenic virus is administered a therapeutically effective amount of anartemisinin-related compound. Oncogenic viruses are a subset of virusesthat include, but is not limited to, HPV, HTLV-1, herpes virus (e.g.,EBV or CMV), SV40-like virus, hepatitis virus, and adenovirus. Theoncogenic virus-induced tumors can be in humans or nonhuman animals. Toillustrate, tumors induced by papillomavirus include lesions at thefollowing sites: cervix, other genital sites (e.g., vagina, penis etc.),anal, oral, upper respiratory, and epidermal. Tumors induced bySV40-like virus include human mesotheliomas, osteosarcomas, and parotidgland tumors. Tumors induced by a herpes virus such as EBV, includenasopharyngeal carcinomas and Hodgkins disease. Tumors induced by HTLV-1include lymphomas. Tumors induced by hepatitis virus includehepatocellular carcinomas.

In another embodiment, the invention provides a method of killing orinhibiting a squamous cell carcinoma in an individual, comprisingadministering to the individual a therapeutically effective amount of anartemisinin-related compound. The squamous cell carcinoma is selectedfrom the group consisting of squamous cell carcinomas of head and neck,oral, oropharyngeal, laryngeal, tracheal, and bronchial. Optionally, thesquamous cell carcinoma contains HPV infection.

In another embodiment, the invention provides a method of killing orinhibiting a squamous cell carcinoma, comprising contacting thecarcinoma with an artemisinin-related compound in an amount sufficientto kill or inhibit growth of carcinoma cells.

In another embodiment, the invention provides a method of inhibitingreplication of a virus in an individual, comprising administering to theindividual a therapeutically effective amount of an artemisinin-relatedcompound. The virus is selected from the group consisting of: humanpapillomavirus (HPV), human T-lymphotropic virus type I (HTLV-1), herpesvirus, SV40-like virus, hepatitis virus, human immunodeficiency virus(HIV), adenovirus, and influenza virus. In some aspects, the virus is anoncogenic virus (e.g., HPV, HTLV-1, herpes virus, SV40-like virus,hepatitis virus or adenovirus). In other aspects, the virus is annon-oncogenic virus (e.g., HIV or influenza virus).

In another embodiment, the invention provides a method of inhibitingreplication of a virus, comprising contacting the virus with anartemisinin-related compound in an amount sufficient to inhibitreplication of the virus.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising an artemisinin-related compound and a secondtherapeutic agent which is not an artemisinin-related compound.Preferably, the second therapeutic agent is a chemotherapeutic agent.

In all embodiments of methods of treating an individual, one or moreartemisinin-related compounds can be administered together(simultaneously) or at different times (sequentially). In addition,artemesinin-related compounds can be administered with another type ortypes of compounds (non-artemisinin compounds). The two types ofcompounds may be administered simultaneously or sequentially.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures of artemisinin and its biologically activemetabolic derivative, dihyroartemisinin (DHA).

FIG. 2 shows that artemisinin is lethal for cervical cancer cells.

FIG. 3 shows that cervical cancer cells, but not normal cervical cells,are efficiently killed by artemisinin.

FIG. 4 shows effect of dihydroartemisinin (DHA) on an EBV positive cellline, the Namalwa cell.

FIG. 5 shows effect of dihydroartemisinin (DHA) on an HTLV-I positivecell line, the MJ cell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on Applicants' discovery thatartemisinin and/or artemisinin derivatives (analogs) are effective inkilling or inhibiting growth of cells that are transformed by humanpapillomavirus (e.g., cervical cancer cells) and cells that aretransformed by other types of virus such as HTLV-1, herpes virus,SV40-like virus, hepatitis virus, HIV, adenovirus or influenza virus. Asdescribed herein, Applicants have shown that artemisinin kills cervicalcancer cells, but not normal cervical cells. Therefore, artemisinin andits derivatives can be used to treat virus infections, and conditionscaused by such virus infections, such as cervical cancers and cervicalprecancers. Artemisinin is currently used in humans as an antimalarialdrug and can be administered both topically and systemically.

In certain embodiments, the invention provides methods of treating anindividual suffering from either a virus infection or a proliferativecervical disorder. As used herein, the individual (patient or subject)to be treated by the subject methods can be either a human or anon-human animal. Such individual is treated by administering to theindividual a therapeutically effective amount of an artemisinin-relatedcompound. As used herein, the term “artemisinin-related compound”includes both artemisinin and artemisinin derivatives or analogs. Theartemisinin derivatives or analogs may be synthetic, semisynthetic ornatural.

In one aspect, the method of the present invention can be used to treatviral infection in an individual caused by human papillomavirus (HPV),human T-lymphotropic virus type I (HTLV-1), herpes virus (e.g., EBV orCMV), SV40-like virus, hepatitis virus, human immunodeficiency virus(H), adenovirus, or influenza virus. The present method can also be usedto treat infections caused by other viruses that are responsive totreatment by artemisinin or artemisinin derivatives, such as DNA virusesand RNA viruses. Such virus may or may not cause cancer.

In another aspect, the present method is a method of treating aproliferative cervical disorder, such as cervical cancer and cervicalprecancer (e.g., cervical dysplasia), in an individual. As used herein,the term “proliferative cervical disorder” refers to anydisease/disorder of the cervix characterized by unwanted or aberrantproliferation of cervical tissue. As one skilled in the art wouldunderstand, the proliferative cervical disorder may be associated with avirus infection such as papillomavirus infection.

In one embodiment, the invention provides a method of killing orinhibiting growth of cells that are infected by human papillomavirus,such as cervical cancer cells, anorectal squamous cancer cells, squamouscarcinoma cells of the skin, basal carcinoma cells of the skin, andsquamous carcinoma cells of the mouth, oropharynx, larynx, head andneck, esophagus, trachea, and bronchi. The infected cells are contactedwith an artemisinin-related compound in a sufficient amount to kill orinhibit growth of infected cells. In this embodiment, a therapeuticallyeffective amount of an artemisinin-related compound is administered toan individual in need of treatment, e.g., for treatment of cervicalcancer, anorectal cancer, squamous carcinoma or basal carcinoma of theskin, and squamous carcinoma of the mouth, oropharynx, larynx, head andneck, esophagus, trachea, and bronchi. The artemisinin-related compoundis administered by a route appropriate for its delivery to the site(s)at which treatment is needed (e.g., topically, intravaginally, rectally,orally, systemically, intramuscularly or intravenously) in an amountsufficient to kill or inhibit growth of cells infected by humanpapillomavirus.

In another embodiment, the invention provides a method of killing orinhibiting growth of cells that are infected by a virus such as HPV,HTLV-1, herpes virus (e.g., EBV or CMV), SV40-like virus, hepatitisvirus, HIV, adenovirus or influenza virus. The infected cells arecontacted with an artemisinin-related compound in a sufficient amount tokill or inhibit growth of the infected cell. In this embodiment, atherapeutically effective amount of an artemisinin-related compound isadministered to an individual in need of treatment of infection by HPV,HTLV-1, herpes virus (e.g., EBV or CMV), SV40-like virus, hepatitisvirus, HIV, adenovirus or influenza virus, by a route which results in(is appropriate for) delivery of an amount sufficient to kill or inhibitgrowth of infected cells to the sites at which treatment is needed.

In one embodiment, the invention provides a method of treating anindividual infected with human papillomavirus (HPV). In this embodiment,a therapeutically effective amount of an artemisinin-related compound isadministered to the individual by a route appropriate for delivery of anamount sufficient to kill or inhibit growth of infected cells to thesite(s) at which treatment is needed. This embodiment is useful to treata variety of conditions in which an individual is infected with HPV,such as conditions in which the cells to be killed or inhibited arecervical cancer cells, anorectal squamous cancer cells, squamouscarcinoma cells or basal carcinoma cells of the skin, and squamouscarcinoma cells of the mouth, oropharynx, and larynx, or in somesquamous cell carcinomas of the head and neck, esophagus, trachea, andbronchi.

In another embodiment, the invention provides a method of treating anindividual infected with a virus, such as HPV, HTLV-1, herpes virus(e.g., EBV or CMV), SV40-like virus, hepatitis virus, HIV, adenovirus orinfluenza virus. In this embodiment, a therapeutically effective amountof an artemisinin-related compound is administered to an individual inneed of treatment of infection by HPV, HTLV-1, herpes virus (e.g., EBVor CMV), SV40-like virus, hepatitis virus, HIV, adenovirus or influenzavirus, by a route which results in (is appropriate for) delivery of anamount sufficient to kill or inhibit growth of infected cells to thesites at which treatment is needed.

In one embodiment, the invention provides a method of treating acondition caused by human papillomavirus in an individual. In thisembodiment, a therapeutically effective amount of an artemisinin-relatedcompound is administered to the individual by a route appropriate fordelivery of an amount sufficient to kill or inhibit growth of infectedcells to the site(s) at which treatment is needed. Conditions caused byHPV that may be treated by the subject methods include, but are notlimited to, cervical cancer, anorectal squamous cancer, squamouscarcinoma or basal carcinoma of the skin, and squamous carcinoma of themouth, oropharynx, larynx, head and neck, esophagus, trachea, andbronchi.

In another embodiment, the invention provides a method of treating acondition caused by a virus in an individual. The virus includes HPV,HTLV-1, herpes virus (e.g., EBV or CMV), SV40-like virus, hepatitisvirus, HIV, adenovirus, and influenza virus. In this embodiment, atherapeutically effective amount of an artemisinin-related compound isadministered to the individual by a route appropriate for delivery of anamount sufficient to kill or inhibit growth of infected cells to thesite(s) at which treatment is needed. Conditions caused by such virusesthat may be treated by the subject methods include, but are not limitedto, cervical cancer, anorectal squamous cancer, squamous carcinoma orbasal carcinoma of the skin, and squamous carcinoma of the mouth,oropharynx, and larynx. Squamous cell carcinomas of the head and neck,esophagus, trachea, and bronchi can also be treated in this manner.

In another embodiment, the invention provides a method of treatingnon-malignant papillomavirus infections, such as benign tumors in anindividual, e.g., laryngeal papillomas, genital papillomas (warts), andcommon warts of hands and feet. The infected cells are contacted with anartemisinin-related compound in an amount sufficient to kill or inhibitgrowth of the cells. For example, a therapeutically effective amount ofan artemisinim-related compound is administered to an individual by aroute which results in delivery of the compound to target site(s) (e.g.,laryngeal tissue, genital warts or common warts of hands and feet) atwhich infected cells are to be killed or inhibited.

In another embodiment, the invention provides a method of treating anindividual suffering from a tumor induced by an oncogenic virus. In thisembodiment, an individual suffering from a tumor which is induced by anoncogenic virus is administered a therapeutically effective amount of anartemisinin-related compound. Oncogenic viruses are a specific subset ofviruses that include, but is not limited to, HPV, HTLV-1, herpes virus(e.g., EBV, CMV, HHV6, or HHV8), SV40-like virus, hepatitis virus, andadenovirus. The oncogenic virus-induced tumors can be in man or inanimals. To illustrate, tumors induced by papillomavirus include lesionsat the following sites: cervix, other genital sites (e.g., vagina, penisetc.), anal, oral, upper respiratory, and epidermal. Tumors induced bySV40-like virus include human mesotheliomas, osteosarcomas, and parotidgland tumors. Tumors induced by a herpes virus such as EBV, includenasopharyngeal carcinomas and Hodgkins disease. Tumors induced byanother herpes virus such as herpes virus type 8 (HHV8), also referredto as KSV, include Kaposi's sarcoma. Kaposi's sarcoma is a malignantcondition and is often diagnosed in immuno-suppressed patients infectedwith HIV. This tumor usually presents as a skin lesion. Tumors inducedby HTLV-1 include lymphomas. Tumors induced by hepatitis virus includehepatocellular carcinomas.

In another embodiment, the invention provides a method of killing orinhibiting a squamous cell carcinoma in an individual, comprisingadministering to the individual a therapeutically effective amount of anartemisinin-related compound. The squamous cell carcinoma is selectedfrom the group consisting of squamous cell carcinomas of head and neck,oral, oropharyngeal, laryngeal, tracheal, and bronchial. Optionally, thesquamous cell carcinoma contains HPV infection.

In another embodiment, the invention provides a method of killing orinhibiting a squamous cell carcinoma, comprising contacting thecarcinoma with an artemisinin-related compound in an amount sufficientto kill or inhibit growth of carcinoma cells.

In another embodiment, the invention provides a method of inhibiting thereplication of a virus in an individual, comprising administering to theindividual a therapeutically effective amount of an artemisinin-relatedcompound. The virus is selected from the group consisting of: humanpapillomavirus (HPV), human T-lymphotropic virus type I (HTLV-1), herpesvirus, SV40-like virus, hepatitis virus, human immunodeficiency virus(HIV), adenovirus, and influenza virus. In some aspects, the virus is anoncogenic virus (e.g., HPV, HTLV-1, herpes virus, SV40-like virus,hepatitis virus or adenovirus). In other aspects, the virus is annon-oncogenic virus (e.g., HIV or influenza virus).

In another embodiment, the invention provides a method of inhibiting thereplication of a virus, comprising contacting the virus with anartemisinin-related compound in an amount sufficient to inhibit thereplication of the virus.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising an artemisinin-related compound and a secondtherapeutic agent which is not an artemisinin-related compound.Preferably, the second therapeutic agent is a chemotherapeutic agent.

Artemisinin-Related Compound.

The term “artemisinin-related compound,” as used herein, refers to bothartemisinin and artemisinin derivatives or analogs. Artemisinin(Qinghaosu) is a naturally occurring substance, obtained by purificationfrom sweet wormwood, Artemisia annua. L. Artemisinin and its analogs aresesquiterpene lactones with a peroxide bridge.

The subject methods contemplate the use of artemisinin derivatives oranalogs. Analogs of artemisinin which have higher solubility in waterare dihydroartemisinin, artemether, artesunate, arteether,propylcarbonate dihydroartemisinin, and artelinic acid.

The very low toxicity of these compounds to humans is a major benefit.Artesunate, for example, is twice as safe as artemether and onlyone-fiftieth as toxic as chloroquinine, the most common antimalarialdrug.

Therapeutically Effective Amount of Artemisinin-Related Compounds

Methods of the present invention comprise administering atherapeutically effective amount of an artemisinin-related compound (oneor more artemisinin-related compound). The phrase “therapeuticallyeffective amount,” as used herein, refers to an amount that results indeath of cells infected by a virus, such as HPV, HTLV-1, herpes virus,SV40-like virus, hepatitis virus, HIV, adenovirus or influenza virus.For example, a therapeutically effective amount of anartemisinin-related compound kills or inhibits growth of cervical cancercells; squamous and basal cell carcinomas of the skin; anorectalsquamous cell carcinomsas; Kaposi's sarcoma, laryngeal papillomas andbenign tumors, such as genital warts and warts of the hands and feet.

Artemisinin is a relatively safe drug and produces few side-effects,even at high doses. Oral doses of 70 mg/kg/day for 6 days have been usedin humans for malaria treatment. Furthermore, more potent analogs ofthis and similar compounds are also available. Higher efficacy ofartemisinin action can be achieved by other means. For example,artemisinin is more reactive with heme than with free iron (Hong et al.,1974, Mol. Biochem. Parasit., 63:121-128). Iron can be introduced intotarget cells using transferrin (see, e.g., Stout et al., 1992, Biochim.Biophy. Res. Comm., 189:765-770) or the heme-carrying compoundhemoplexin (see, e.g., Smith et al., 1988, Biochem. J., 256:941-950;Smith et al., 1990, Europ. J. Cell Biol., 53:234-245). Theconcentrations of agents for enhancing intracellular iron concentrationsin the practice of the present invention will generally range up to themaximally tolerated dose for a particular subject and agent, which willvary depending on the agent, subject, disease condition and otherfactors. Dosages ranging from about 1 to about 100 mg of iron perkilogram of subject body weight per day will generally be useful forthis purpose.

The dose of artemisinin or artemisinin derivative compounds administeredto an individual in need of treatment will vary and will be determinedfor each individual with reference to, for example, the compound used,the route of administration, and the physical condition and body size ofthe individual. To illustrate, about 0.1 to about 100 mg per kilogram ofbody weight per day can be administered. In further embodiments, fromabout 1 to about 90 mg per kilogram of body weight per day isadministered. Alternatively, from about 1 to about 75 mg per kilogram ofbody weight per day can be administered. The daily dosage may beadministered as a single dosage or may be divided into multiple doses.

Actual dosage levels of the artemisinin-related compound may be variedso as to obtain amounts at the site of target cells (e.g., virusinfected cells or abnormal cervical cells), effective to obtain thedesired therapeutic or prophylactic response. Accordingly, the selecteddosage level will depend on the nature and site of the target cells, thedesired quantity of artemisinin-related compound required at the targetcells for inhibition or killing, the nature of the artemisinin-relatedcompound employed, the route of administration, and other factors.Topical or oral administration, for instance, may typically be carriedout one or more times a day, such as one to three times daily.

Pharmaceutical Compositions.

In certain embodiments of methods of the present invention,artemisinin-related compounds are formulated with a pharmaceuticallyacceptable carrier.

Artemisinin or an artemisinin derivative can be administered alone or asa component of a pharmaceutical formulation (composition). The compoundsmay be formulated for administration in any convenient way for use inhuman or veterinary medicine. In certain embodiments, the compoundincluded in the pharmaceutical preparation may itself be active, or maybe a prodrug. The term “prodrug” refers to compounds which, underphysiological conditions, are converted into therapeutically activeagents.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Formulations of the artemisinin-related compounds include those suitablefor oral/nasal, topical, parenteral, intravaginal and/or rectaladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect.

Methods of preparing these formulations or compositions includecombining an artemisinin-related compound and a carrier and, optionally,one or more accessory ingredients. In general, the formulations areprepared by combining an artemisinin-related compound with a liquidcarrier, or a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Formulations of the artemisinin-related compounds suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofan artemisinin-related compound as an active ingredient. Anartemisinin-related compound may also be administered as a bolus,electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), an artemisinin-relatedcompound is mixed with one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate, and/or any of thefollowing: (1) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

Liquid dosage forms for oral administration of the artemisinin-relatedcompounds include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as water or other solvents, solubilizing agentsand emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming, and preservativeagents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In particular, methods of the invention can be administered topically,either to skin or to mucosal membranes such as those on the cervix andvagina. This offers the greatest opportunity for direct delivery totumor with the lowest chance of inducing side effects. The topicalformulations may further include one or more of the wide variety ofagents known to be effective as skin or stratum corneum penetrationenhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone,dimethylacetamide, dimethylformamide, propylene glycol, methyl orisopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents mayfurther be included to make the formulation cosmetically acceptable.Examples of these are fats, waxes, oils, dyes, fragrances,preservatives, stabilizers, and surface active agents. Keratolyticagents such as those known in the art may also be included. Examples aresalicylic acid and sulfur.

Dosage forms for the topical or transdermal administration of anartemisinin-related compound include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches, and inhalants. The activecompound may be mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantswhich may be required. The ointments, pastes, creams and gels maycontain, in addition to an artemisinin-related compound, excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an artemisinin-relatedcompound, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more artemisinin-related compounds in combination withone or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe artemisinin-related compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Formulations of the artemisinin-related compounds for intravaginaladministration may be presented as a suppository, which may be preparedby mixing one or more compounds of the invention with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound. Optionally, such formulations suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

In another embodiment, artemisinin or artemisinin derivative compoundscan be administered to animals in animal feed. For example, thesecompounds can be included in an appropriate feed premix, which is thenincorporated into the complete ration in a quantity sufficient toprovide a therapeutically effective amount to the animal. Alternatively,an intermediate concentrate or feed supplement containing theartemisinin-related compounds can be blended into the feed. The way inwhich such feed premixes and complete rations can be prepared andadministered are described in reference books (see, e.g., “AppliedAnimal Nutrition,” W. H. Freedman and CO., San Francisco, U.S.A., 1969or “Livestock Feeds and Feeding,” O and B books, Corvallis, Oreg.,U.S.A., 1977).

Methods of Administration.

In certain embodiments, the subject methods of the invention can be usedalone. Alternatively, the subject methods may be used in combinationwith other anti-viral or anti-cancer therapeutic approaches (e.g.,administration of an anti-viral or anti-cancer agent, radiation therapy,phototherapy or immunotherapy) directed to treatment or prevention ofproliferative cervical disorders or virus infections. For example, suchmethods can be used in prophylactic cancer prevention, prevention ofcancer recurrence and metastases after surgery, and as an adjuvant ofother traditional cancer therapy. Similarly, the subject methods of theinvention may be combined with other antiviral therapies.

Thus, the subject methods of the invention may further include asoptional ingredients one or more agents already known for their use inthe inhibition of cervical cancer or precancer cells, for added clinicalefficacy. These agents include, but are not limited to,interleukin-2,5′-fluorouracil, nedaplatin, methotrexate, vinblastine,doxorubicin, carboplatin, paclitaxel (Taxol), cisplatin, 13-cis retinoicacid, pyrazoloacridine, and vinorelbine. Appropriate amounts in eachcase will vary with the particular agent, and will be either readilyknown to those skilled in the art or readily determinable by routineexperimentation. methotrexate, vinblastine, doxorubicin, and cisplatin.

In other cases, the subject methods of the invention may further includeas optional ingredients one or more agents already known for theiranti-viral effects, for added clinical efficacy. These agents include,but are not limited to, 5′-fluorouracil, interferon alpha, imiquimod,lamivudine, arsenic trioxide, capsaicin, nucleoside analogues (e.g.,acyclovir), and antiviral vaccines.

The artemisinin-related compounds may be employed in vitro, in vivo orex vivo for killing or inhibition of affected cells. For in vivoapplications, artemisinin-related compounds can be administrated to ahuman or other animal subject, together with a pharmaceuticallyacceptable carrier, to localize a sufficient amount at target tissuesites to facilitate killing or inhibition of target cells.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Effect of Artemisinin and its Analogs on Cervical Cancer Cells

FIG. 2 shows that artemisinin is lethal for cervical cancer cells. Theindicated cervical cancer cell lines were treated with 25 μM artemisinin(or control solvent) for 3 days and then photographed with a phasecontrast microscope. Normal cervical cells (HCX) showed little change inmorphology in response to artemisinin whereas the cervical cancer cellsrounded up and detached from the tissue culture plate.

FIG. 3 shows that cervical cancer cells, but not normal cervical cells,are efficiently killed by artemisinin. A dose-response curve is shownfor the effects of dihydroartemisinin (DHA) on the viability of normalcervical cells (HCX) and 3 cervical cancer cell lines (HeLa, SiHa,Caski). The cervical cancer cell lines demonstrated 80% loss ofviability within 3 days of treatment with 25 μM DHA. HeLa cells were themost sensitive, exhibiting 95% cell death at 25 μM DHA.

Example 2 Effect of Dihydroartemisinin (DHA) on Virally TransformedLymphoid Cell Lines

Applicants carried out studies to evaluate the in vitro effect ofartemisinin (DHA) on two virally transformed lymphoid cell lines. Onecell line, referred to as MJ, is an HTLV-I positive cutneous T cellleukemia line and the other cell line, referred to Namalwa, is an EBVpositive Burkitt's lymphoma B cell line.

The cell lines were maintained in culture, using RPMI 1640 medium,supplemented with 10% fetal bovine serum and antibiotics. For the assay,100,000 cells, in volumes of 100 μl medium, were placed in micro-titerwells; an additional 100 μl medium, containing 0 μM, 6.25 μM, 12.5 μM,25 μM, 50 μM, 100 μM and 200 μM concentrations of DHA, were added. Thenegative controls contained medium only. The DHA stock (20 mM) was usedto dilute the drug in concentrations ranging from 0 to 200 μM. Allexperiments were performed in triplicate. After incubation at 37° C. and5% CO₂, for various time periods, the cells were counted in ahemocytometer in the presence of trypan blue. Viability was expressed,as % of controls (no drug).

As shown in FIG. 4 and in FIG. 5, DHA killed approximately 60% of bothcell types (MJ and Namalwa), at concentrations of 6.25 μM. DHA may killthe transformed cells by interfering with viral mechanisms. Therefore,artemisinin and its analogs (derivatives) may have antiviral activity(including anti-retroviral activity against HTLV and HIV).

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. A method of treating an individual suffering from a virus infection,comprising administering to the individual a therapeutically effectiveamount of an artemisinin-related compound.
 2. The method of claim 1,wherein the artemisinin-related compound is artemisinin.
 3. (canceled)4. The method of claim 1, wherein the virus is selected from the groupconsisting of: human papillomavirus (HPV), human T-lymphotropic virustype I (HTLV-1), herpes virus, SV40-like virus, hepatitis virus, humanimmunodeficiency virus (HIV), adenovirus, and influenza virus.
 5. Themethod of claim 4, wherein the herpes virus is Epstein-Barr virus (EBV),cytomegalovirus (CMV), or herpes virus 8 (HHV8).
 6. The method of claim1, wherein the artemisinin-related compound is administered by a routeselected from the group consisting of oral administration, topicaladministration, parenteral administration, intravaginal administration,rectal administration, systemical administration, intramuscularadministration, and intravenous administration.
 7. The method of claim1, wherein the artemisinin-related compound is formulated with apharmaceutically acceptable carrier.
 8. The method of claim 1, whereinthe artemisinin-related compound is administered with an anti-viralagent.
 9. The method of claim 8, wherein the artemisinin-relatedcompound and the anti-viral agent are in the same formulation.
 10. Amethod of treating an individual suffering from a proliferative cervicaldisorder, comprising administering to the individual a therapeuticallyeffective amount of an artemisinin-related compound.
 11. The method ofclaim 10, wherein the artemisinin-related compound is artemisinin. 12.(canceled)
 13. The method of claim 10, wherein the proliferativecervical disorder is cervical cancer.
 14. The method of claim 10,wherein the proliferative cervical disorder is cervical dysplasia. 15.The method of claim 10, wherein the artemisinin-related compound isadministered by a route of oral administration, topical administration,parenteral administration, intravaginal administration, systemicaladministration, intramuscular administration or intravenousadministration.
 16. The method of claim 10, wherein theartemisinin-related compound is formulated with a pharmaceuticallyacceptable carrier.
 17. The method of claim 10, wherein theartemisinin-related compound is combined with an anti-cancer therapy.18. The method of claim 17, wherein the artemisinin-related compound andthe anti-cancer agent are in the same formulation.
 19. The method ofclaim 10, wherein the proliferative cervical disorder is associated withpapillomavirus infection.
 20. A method of killing or inhibiting growthof cells that are infected by human papillomavirus, comprisingcontacting the cells with an artemisinin-related compound in an amountsufficient to kill or inhibit growth of infected cells.
 21. The methodof claim 20, wherein the cells are human cells.
 22. The method of claim21, wherein the human cells are selected from the group consisting of:cervical cancer cells, anorectal squamous cancer cells, squamouscarcinoma cells of the skin, basal carcinoma cells of the skin, andsquamous cell carcinoma cells of the mouth, oropharynx, larynx, head andneck, esophagus, trachea, and brochi. 23-25. (canceled)
 26. A method oftreating an individual infected with human papillomavirus, comprisingadministering to the individual a therapeutically effective amount of anartemisinin-related compound, wherein the artemisinin-related compoundselectively kills or inhibits growth of papillomavirus infected cells.27. The method of claim 26, wherein the cells are human cells.
 28. Themethod of claim 27, wherein the human cells are selected from the groupconsisting of: cervical cancer cells, anorectal squamous cancer cells,squamous carcinoma cells of the skin, basal carcinoma cells of the skin,and squamous carcinoma cells of the mouth, oropharynx, larynx, head andneck, esophagus, trachea, and bronchi. 29-31. (canceled)
 32. A method oftreating a condition caused by human papillomavirus in an individual,comprising administering to the individual an artemisinin-relatedcompound in a therapeutically effective amount. 33-34. (canceled)
 35. Amethod of killing or inhibiting growth of non-malignant HPV infectedcells in an individual, comprising contacting the cells with anartemisinin-related compound in an amount sufficient to kill or inhibitgrowth of the cells.
 36. The method of claim 35, wherein the cells areselected from the group consisting of: cells of laryngeal papilloma,cells of genital papillomas (warts), and cells of common warts of handsand feet. 37-41. (canceled)
 42. A method of killing or inhibiting asquamous cell carcinoma in an individual, comprising administering tothe individual a therapeutically effective amount of anartemisinin-related compound.
 43. The method of claim 42, wherein thesquamous cell carcinoma is selected from the group consisting ofsquamous cell carcinomas of head and neck, oral, oropharyngeal,laryngeal, tracheal, and bronchial.
 44. The method of claim 42, whereinthe squamous cell carcinoma contains HPV infection. 45-54. (canceled)